肺炎链球菌重组酶聚合酶扩增检测方法的建立与应用  

Establishment and application of recombinase polymerase amplification in the detection of Streptococcus pneumoniae

作　　者：章太婵 廖佳宇 韦华贵 梁雪雁 林敏[2,3] 陈江涛 ZHANG Taichan;LIAO Jiayu;WEI Huagui;LIANG Xueyan;LIN Min;CHEN Jiangtao(Guangdong Medical University,Zhanjiang,Guangdong,524000,China;Department of Laboratory,Chaozhou People's Hospital Affiliated to Shantou University,Chaozhou,Guangdong,521000,China;Department of Laboratory,The Affiliated Hospital of Youjiang Medical University for Nationalities,Baise,Guangxi,533000,China;Department of Laboratory,Huizhou Central People's Hospital Affiliated to Guangdong Medical University,Huizhou,Guangdong,516000,China)

机构地区：[1]广东医科大学,广东湛江524000 [2]汕头大学附属潮州市人民医院检验科,广东潮州521000 [3]右江民族医学院附属医院检验科,广西百色533000 [4]广东医科大学附属惠州市中心人民医院检验科,广东惠州516000

出　　处：《当代医学》2024年第34期68-72,共5页Contemporary Medicine

基　　金：惠州市科技研发计划社会发展领域研发项目(210426104574869)。

摘　　要：目的建立一种重组酶聚合酶扩增(recombinase polymerase amplification,RPA)检测方法,用于临床快速检测肺炎链球菌(Streptococcus pneumoniae,S.pn),并对该研究的灵敏度、特异度及临床样本中S.pn的检出率进行评价。方法根据美国国家生物技术信息中心中S.pn自溶素(autolysin,LytA)基因序列设计3对RPA引物,采用RPA试剂筛选出最优RPA扩增引物对,从而建立S.pn的RPA检测方法,用该方法检测临床标本的阳性率,并与常规PCR、细菌分离培养的检出率对比,评价该方法的实用性。结果RPA技术检测S.pn的灵敏度为10拷贝/μl,与常规PCR一致,且特异度高,其他致病菌均未检测到阳性信号。此外,对于临床送检的标本RPA技术阳性检出率为40.00%,与常规PCR方法检出率无差异,细菌分离培养的阳性检出率为41.43%,而RPA技术与常规PCR方法与细菌分离培养阳性符合率均一致为100.00%。结论S.pn RPA检测方法具有高效及灵敏、特异度强等特点,可为临床及现场快速检测S.pn疾病提供了新的方向。Objective To establish a method of recombinase polymerase amplification(RPA)to rapidly identify Streptococcus pneumoniae(S.pn)in clinic,and assess its sensitivity,specificity and rate of detection of S.pn in clinical samples.Methods According to the sequence of S.pn autolysin gene(LytA)in National Center for Biotechnology Information,three pairs of RPA primers were designed,and the optimal RPA amplification primer pair was screened by RPA reagent,so as to establish the RPA detection method of S.pn.Finally,the positive rate of clinical specimens was detected by this method,and compared with the detection rate of conventional PCR and bacterial isolation and culture to evaluate the practicability of this method.Results The sensitivity of RPA to detect S.pn was 10 copies/μl,which was consistent with conventional PCR,and the specificity was high,and no positive signal was detected for other pathogenic bacteria.Additionally,the positive detection rate of RPA technology for clinical specimens was 40.00%,which was no different from that of conventional PCR method,and the positive detection rate of bacterial isolation culture was 41.43%,while the positive coincidence rate of both RPA technology and conventional PCR method with bacterial isolation culture was 100.00%.Conclusion The detection method of S.pn RPA has the characteristics of high efficiency,sensitivity and specificity,which can provide a new direction for the rapid detection of S.pn disease in clinic and in the field.

关 键 词：肺炎链球菌 自溶素基因 重组酶聚合酶扩增 快速检测 

分 类 号：R44[医药卫生—诊断学]